More than fifty different species of Plasmodium can cause malaria in humans, monkeys, birds, fish, cattle and rodents. The development of diagnostic assays for the detection of Plasmodium in humans and animals is therefore highly desirable.
Human malaria, which is caused by Plasmodium ssp., in particular P. falciparum, P. vivax, P. malariae, and P. ovale, remains one of the major health problems around the world.
Plasmodium vivax induces a moderate form of malaria, vivax malaria, characterized by periodic chills and fever, an enlarged spleen, anaemia, severe abdominal pain and headaches, and extreme lethargy. If left untreated, the disease tends to be self-limiting within a period of 10 to 30 days, but will recur periodically. Although the fatality rate of vivax malaria is low, the disease is highly debilitating and makes the patient more vulnerable to other diseases.
The incubation period ranges from 10 days to 4 weeks. Generally, paroxysms of chills and fever appear on the 14th day after the bite of an infected female anopheles mosquito. During this time the parasite has been multiplying in the liver cells of the patient. Paroxysms continue to recur every other day, as the parasite completes its 48-hour cycle of development, now in the blood. During the paroxysm, the patient first goes through a “cold stage” during which he has chilly sensations, his skin is blue, his teeth chatter and there is violent shaking. After an hour, the “hot stage” is ushered in, with a rise in temperature to as high as 107° F. (41.7° C.); the skin is hot and dry and the patient complains of severe headache. The fever lasts about 2 hours, and is followed by the “sweating stage”, during which there is profuse perspiration, the temperature falls to normal, the headache disappears, and although weak and drowsy, the patient feels well.
Plasmodium ovale produces a disease very similar to vivax malaria.
Plasmodium malariae, the causative agent of quartan malaria, has an incubation period of 18-40 days. The paroxysms occur every 72 hours, and are longer and somewhat more severe than those accompanying vivax malaria.
Plasmodium falciparum-induced malaria (falciparum malaria) presents oedema of the brain and lungs and blockage of the kidneys, in addition to the symptoms associated with vivax malaria. Unless treated promptly, the fatality rate of falciparum malaria is high, especially in juveniles.
Paroxysms associated with falciparum malaria occur irregularly after a 12-day incubation period. They are severe, and accompanied by high temperatures. The so-called cerebral algid, haemorrhagic and pernicious types of malaria represent forms of falciparum malaria with different localizations of the parasite. In the cerebral type, the onset is delirium and coma, and death may occur in several hours without return to consciousness. “Black-water fever” or haemorrhagic malaria is a type in which haemolysis or dissolution of the red cells occurs, and dark urine due to the presence of haemoglobin is an outstanding feature. In the algid form, there are vomiting, diarrhea, and subnormal temperature.
The life cycle of the parasite and its course in the human body proceeds in the following way. The saliva of the mosquito contains the Plasmodium at the lance-shaped sporozoite stage of its life cycle. Upon inoculation of the host by biting, the sporozoites quickly migrate to the liver where they divide and develop into multi nucleated schizonts. Within 6 to 12 days, the schizonts disrupt and release into the blood the form known as merozoites. Each liver cell infected by one sporozoite releases into the blood stream from 10,000 to 30,000 merozoites. These later invade the host□s erythrocytes where they grow and form more schizonts which, in turn, again divide, releasing more merozoites into the blood stream to repeat the cycle. The principal symptoms of malaria are associated with the rupture of the schizonts, the periodic lysis of the blood cells with release of merozoites and toxic wastes which cause the regular fevers and chills of malaria.
Neither vector control measures nor immuno or chemoprophylaxis have proven effective in eradicating the disease. Thus, more than ever, chemotherapy appears to be crucial in dealing with both the prevention and treatment of malaria. However, presently used drugs are constantly losing their efficacy due to the development of drug resistance by the parasite. For example, drug resistance of Plasmodium falciparum to chloroquine has occurred in Bangladesh, Brazil, Burma, Colombia, Ecuador, Guyana (French), Guyana, India, Indonesia, Kampuchea, Malaysia, Nepal, Pakistan, Panama, Philippines, Surinam, Thailand, Venezuela, and Vietnam, amongst others. Therefore, the design of novel drugs is urgent.
Targets for drug design are generally nuclear-encoded gene products. However, inter-specific and developmental variation in nuclear gene expression has reduced the general efficacy of drugs which target such nuclear-encoded gene products.
Diagnosis of malaria is generally made by microscopic examination of blood films taken during episodes of fever, when the parasites may be seen. In general, the Plasmodium parasite is detected microscopically by examining finger prick blood samples for the presence of the morphologically distinct parasite using Giemsa stain solution (Shute et al., 1980). This needs to be done by an experienced microscopist since Plasmodium falciparum and Plasmodium vivax are morphologically similar, albeit not identical. In view of the distinct epidemiologies of P. falciparum compared to P. vivax, it is important that diagnosis of infection by these species have a low error rate. Any incorrect diagnosis of falciparum malaria, for example, may be fatal for the patient. The microscopic technique is limited in so far as the method is slow and specialised personnel is required to perform the technique.
A variation of the standard microscopic assay, the quantitative buffy coat (QBC) technique is based upon the ability of parasite nucleoproteins to absorb acridine orange and fluoresce (Wardlaw et al, 1983). The fluorescent nucleoproteins are readily visible against a background of non-fluorescent red blood cells. Although the method is more sensitive than the standard microscopic assay, it suffers from the disadvantages associated with the standard microscopic assay. Furthermore, the requirement of costly fluorescence microscopes and centrifuges to perform the QBC assay, renders the method unrealistic in resource-limited settings which often lack even electricity.
Immunological tests, for example the ParaSight™ F test (Becton Dickinson) and the similar ICT Malaria P.f. test (ICT Diagnostics) detect the Plasmodium falciparum histidine-rich protein HRP2 in blood samples derived from patients. A major drawback associated with such methods is that they require Plasmodium falciparum gene expression to occur before the organism can be detected. Furthermore, as considerable variation in gene expression can occur between Plasmodium ssp., these tests tend to be species-specific. For example, the ParaSight™ F test (Becton Dickinson) and ICT Malaria P.f. test (ICT Diagnostics) are specific for Plasmodium falciparum only and incapable of detecting other species. Furthermore, these tests, in particular the ParaSight™ F test (Becton Dickinson), are subject to a high proportion of false-negative detections, such that a higher than acceptable frequency of patients infected with a Plasmodium ssp. go undetected.
Immunological techniques such as the enzyme-linked immunosorbent assay (ELISA) or the radio immunoassay (RIA) which detect genus- and species-specific parasite antigens also exist. However, such methods are constrained by immunological cross-reaction between parasite and host antigens on the one hand and between parasite antigens and antigens derived from other microorganisms on the other hand. As a consequence, the susceptibility of immunological methods to false positive detection of Plasmodium is high. As already mentioned above, species-specific detection methods lead to a large number of false-negative detections.
Furthermore, as different Plasmodium antigens are expressed at different developmental stages, immunological techniques may only detect the parasite at certain stages of development. Such antigenic diversity displayed by Plasmodium is a major obstacle to the application of immunological techniques. In addition, radioisotope-based assays such as the RIA are impractical for field use. Immunological methods cannot distinguish between past and present infections.
State-of-the art diagnostic assays, which rely on the detection of Plasmodium genomic DNA in a sample, are species-specific and not capable of general application for any Plasmodium ssp., in part because there is considerable variation in genomic DNA between Plasmodium species, such variation precluding the simultaneous detection of several Plasmodium ssp. in a single biological sample or alternatively, the use of a single DNA-based assay for the detection of any Plasmodium ssp. in a biological sample derived from a human or animal subject suspected of carrying the parasite.
As a consequence of the foregoing, there is a high demand for a reliable and simple technology for the diagnosis of Plasmodium in human and animal tissues.
Plasmodium ssp. possess additional genomes with potentially crucial functions (Wilson et al., 1991). Until the present invention, very little was known about this extrachromosomal material. Furthermore, the function of the extrachromosomal plastid element in the protozoans remains to be determined. To date, there is no clear evidence for DNA replication or functionally active gene products from the plastid element.